SUMMARY The objective of this proposal is to develop a label-free device that can sort single cells based on metabolism. Glycolysis plays a fundamental role in cell signaling, activation, proliferation and disease progression. Shifts in metabolism from oxidative phosphorylation to glycolysis have been linked to such processes as the activation of T cells and the reprograming and proliferation of stem cells. Many disease states, including diabetes, cancer, obesity and metabolic disorders involve altered glycolysis. Conventional techniques are limited to the measurement of glycolysis via fluorescence assays or extracellular acidification. The isolation of live single cells based on metabolism would allow the analysis (genomics, proteomics, metabolomics, etc.), culture and targeting of aberrant cells. Our device will allow the high-throughput sorting of live cells based on glycolytic activity in a microfluidic device without the use of any labels, markers or active sorting components. In the first aim, the technique will be used to separate live and dead cells with accuracy and high-throughput. This platform can serve as a free-standing, inexpensive technology to quickly and easily separate live and dead cells for applications in pharmacology and toxicology. In the second aim of the proposal, the device will be used to isolate cells with high glycolytic activity that will be analyzed by single-cell whole transcriptome analysis and compared to the larger population of cultured cells. This analysis will reveal the cell-to-cell heterogeneity in gene expression and clarify the determinants of highly glycolytic cells. Single- cell gene expression may also reveal contributors to glycolytic activity that may be masked in bulk studies. The developed technology can find broad use for the isolation of rare cells or subpopulations with high rates of metabolism for applications in medicine, cell biology, bioenergy, biotechnology and studying the microbiome.